Chemotherapy still plays an important role in cancer treatment today even though medical oncology has developed. In particular, chemotherapy is mainly employed in treatment of leukemia, and provides a higher therapeutic effect against leukemia than that against solid cancer. Especially, acute myeloid leukemia (AML) cells are known to be highly sensitive to drugs. For this reason, when remission-induction therapy is applied as treatment of AML, complete remission is observed in about 80% of cases. However, recurrence is observed in many cases, and a long-term survival rate achieved today is only from 30% to 40%. It is thought that recurrence is caused by a small number of leukemia cells remaining in the bone marrow of patients in a complete remission period after chemotherapy.
Leukemia cells in the non-adhered state in peripheral blood or body fluid are killed by anticancer agents with relatively high efficiency. However, it is known that leukemia cells in bone marrow adhere to extracellular matrixes (such as fibronectin (FN) or marrow stromal cells) via adhesion molecules (such as integrin), and thereby acquire resistance to anticancer agents, as a result of which a small number of leukemia cells remain even after chemotherapy. This phenomenon is called cell adhesion mediated drug resistance (CAM-DR), which is one of the most important problems to be solved in treatment of leukemia using anticancer agents.
Fibronectin is involved in adhesion and attachment-detachment of cells. Fibronectin is one of representative extracellular matrix protein molecules. Fibronectin is distributed in almost all tissues, and serves as a framework for building a tissue, as well as binds to adhesion molecules on the cell membrane and acts as a signal molecule for regulating cell functions.
A polypeptide constituting a fibronectin molecule is composed of type I, type II, and type III repetitive sequences. Among them, a fibronectin (FN) type III repeat (FNIII) is known to have various functions.
For example, FNIII is known to have a cell adhesion inhibitory activity (for example, see Japanese Patent Application Laid-Open (JP-A) No. H10-147600 and JP-A No. 2000-26490). JP-A No. H10-147600 mentions the possibility of an effect in terms of suppressing metastasis of cancer, but does not describe any working examples thereof. International Publication No. (WO) 01/08698 discloses an effect in terms of enhancing an anticancer activity based on the apoptosis induction activity of FNIII. However, the working examples disclosed in WO 01/08698 describe only the results of in vitro experiments, and does not study in vivo efficacy; therefore, there is no information about stability in blood and the like.
Furthermore, WO 01/08698 discloses only in vitro experiment, and includes no disclosure about effects in living organisms. Accordingly, practical judgment cannot be made with respect to an increase and enhancement of anticancer activity based on the apoptosis induction activity of FNIII.
Specifically, in FIGS. 1 to 8 of WO 01/08698, the effect achieved by mixing vincristine or actinomycin D with a polypeptide represented by SEQ ID NO: 2 is investigated using some cancer cells. However, data indicating practical hopefulness are not demonstrated; for example, the effect achieved by the addition is about 30% at most in FIGS. 1, 2, 3, 4, and 8, and an opposite effect is obtained depending on the concentration as in FIG. 2. Although FIGS. 5 and 7 illustrate an effect achieved by addition of the agent, the effect achieved by the agent itself does not show concentration dependence, and, therefore, the efficacy thereof cannot be evaluated. Only FIG. 6 suggests a tendency that the addition of polypeptide represented by SEQ ID NO: 2 exerts an effect on vincristine in a case in which GT3TKB cells derived from stomach cancer are used.
As is understood from the above, in consideration of clinical use in practice, it is thought that the addition of the polypeptide represented by SEQ ID NO: 2 exerts an effect only on a specific kind of cancer and a specific agent, and that the polypeptide represented by SEQ ID NO: 2 does not enhance the effects of all anticancer agents. Moreover, an effect achieved by using the polypeptide represented by SEQ ID NO: 2 with cytarabine or the like, which is a pyrimidine antimetabolite having another mechanism of action among anticancer agents, is not demonstrated.
JP-A No. 2006-327980 describes working examples which aim to cause remission in mice by using an antileukemic agent and a FNIII-like peptide in combination in animal experiments. However, the concentration of the FNIII-like peptide in the working examples is 1 mg, and no effect is confirmed at concentrations lower than this concentration.
In addition, experiments similar to those of JP-A No. 2006-327980 are reported in Leukemia (2008), Vol. 22, 353-360. However, this report demonstrates that even a modified FNIII-like peptide exhibits no improvement in physiological activity.
There are some reports about such a chemical modification. JP-A No. 2010-043087 reports that even when native HBHA was produced by genetic recombination in E. coli, the physiological activity thereof was different from that of HBHA produced by Mycobacterium, but inherent physiological activity thereof could be reproduced by methylation of a lysine group at the C-terminal. This merely indicates that the methylation led to recovery of the inherent activity, and does not demonstrate a higher effect than the inherent physiological activity. JP-A No. H6-73093 reports examples in which D-form amino acids such as Trp, His, or Phe were introduced for the purpose of eliminating defects of opioid analgesics such as dependence and tolerance. However, as described in JP-A No. H6-73093, these examples are not examples that achieved improvement in stability and activity as a compound. In addition, although one may think that the use of D-form amino acids prevents decomposition due to general inability of proteases to recognize D-form amino acids, it is also thought that the physiological activity of the peptide cannot be maintained due to a changed conformation of the obtained peptide. Biochem Pharmcol. 1999 Dec. 1; 58(11); 1775-80 reports that replacement of two residues at each of N-terminal and C-terminal with D-form amino acids resulted in increased stability in serum enzyme treatment, but did not increase the physiological activity. In Neuropeptides, 1984 December; 5(1-3): 177-80, it is reported that, for example, modification of enkephalin (Tyr-Gly-Gly-Phe-Leu (SEQ ID NO: 2)) from among the opioid peptide group to Tyr-D-Ala-Gly-Phe-Leu (SEQ ID NO: 4) by replacement of the second amino acid did not increase the activity.